The LIM and POU Homeobox Genes Ttx-3 and Unc-86 Act As Terminal

RESEARCH ARTICLE

The LIM and POU homeobox genes ttx-3 and unc-86 act as terminal selectors in distinct cholinergic and serotonergic neuron types Feifan Zhang1, Abhishek Bhattacharya1, Jessica C. Nelson2, Namiko Abe1, Patricia Gordon1, Carla Lloret-Fernandez3, Miren Maicas3, Nuria Flames1,3, Richard S. Mann1, Daniel A. Colón-Ramos2 and Oliver Hobert1,4,*

ABSTRACT factor initiates and maintains the terminal differentiation program of Transcription factors that drive neuron type-specific terminal dopaminergic neurons in the midbrain (Smidt and Burbach, 2009), differentiation programs in the developing nervous system are often whereas the Pet1 transcription factor initiates and maintains the expressed in several distinct neuronal cell types, but to what extent terminal differentiation program of serotonergic neurons (Liu et al., they have similar or distinct activities in individual neuronal cell types 2010). However, few neuronal transcription factors are expressed is generally not well explored. We investigate this problem using, as exclusively in only one specific neuronal cell type (Gray et al., 2004; a starting point, the C. elegans LIM homeodomain transcription factor Lein et al., 2007). For example, in addition to being expressed in ttx-3, which acts as a terminal selector to drive the terminal midbrain dopaminergic neurons, Nurr1 is expressed in other non- differentiation program of the cholinergic AIY interneuron class. Using dopaminergic neuronal cell types in which its function is not well a panel of different terminal differentiation markers, including understood, such as the adult olfactory bulb, specific cortical areas neurotransmitter synthesizing enzymes, neurotransmitter receptors and the hippocampus (Zetterström et al., 1996). The expression of a and neuropeptides, we show that ttx-3 also controls the terminal given transcription factor in distinct neuronal populations poses the differentiation program of two additional, distinct neuron types, fundamental question of whether there are underlying common namely the cholinergic AIA interneurons and the serotonergic NSM themes in the activity of the transcription factor in distinct neuronal neurons. We show that the type of differentiation program that is cell types. controlled by ttx-3 in different neuron types is specified by a distinct We have undertaken a systematic, in-depth comparison of the set of collaborating transcription factors. One of the collaborating activity of two transcription factors in the development of several transcription factors is the POU homeobox gene unc-86, which distinct neuronal cell types in the nematode C. elegans, examining collaborates with ttx-3 to determine the identity of the serotonergic whether there are indeed conceptual similarities in the activities of NSM neurons. unc-86 in turn operates independently of ttx-3 in the a given transcription factor in distinct neuron types. We used, as a anterior ganglion where it collaborates with the ARID-type starting point, a member of the LIM homeobox gene family, an transcription factor cfi-1 to determine the cholinergic identity of the ancient family of neuronal patterning genes that display complex IL2 sensory and URA motor neurons. In conclusion, transcription expression patterns in the nervous system of many different species, factors operate as terminal selectors in distinct combinations in from invertebrates to vertebrates (Hobert and Westphal, 2000; different neuron types, defining neuron type-specific identity features. Simmons et al., 2012; Srivastava et al., 2010). One unifying theme is their expression in terminally differentiating neurons (Hobert and KEY WORDS: Caenorhabditis elegans, Homeobox, Neuron Ruvkun, 1998; Moreno et al., 2005). We focus here on the ttx-3 LIM differentiation homeobox gene, which is the sole C. elegans member of the Lhx2/9 subclass of LIM homeobox genes. In vertebrates, Lhx2 is expressed INTRODUCTION in multiple neuronal cell types and is required for the differentiation The development of the nervous system is a multistep process that of olfactory sensory neurons (Hirota and Mombaerts, 2004; employs a series of sequentially acting regulatory factors that Kolterud et al., 2004), the specification of cortical neuron fate successively restrict and determine cellular fates. During the process (Mangale et al., 2008) and the differentiation of thalamic neurons of terminal differentiation, individual neuron types acquire specific, (Peukert et al., 2011). Whether there is a common theme in the hard-wired features that are maintained by the neuron type function of Lhx2 in these distinct neuronal cell types is not known. throughout the life of the animal. A number of transcription factors The C. elegans Lhx2/9 ortholog ttx-3 is exclusively expressed in have been identified that initiate and maintain specific terminal a small number of neurons in distinct head ganglia (Altun-Gultekin differentiation programs in the developing nervous system (Hobert, et al., 2001). ttx-3 null animals display broad differentiation defects 2011). For example, in mouse, the Nurr1 (Nr4a2) transcription in the cholinergic AIY interneuron class. AIY interneurons of ttx-3 null mutants are generated and still express pan-neuronal features, 1Department of Biochemistry and Molecular Biophysics, Columbia University but fail to express scores of terminal identity markers that define the 2 Medical Center, New York, NY 10032, USA. Department of Cell Biology, Yale functional properties of AIY, including genes required to synthesize University School of Medicine, New Haven, CT 06520, USA. 3Instituto de Biomedicina de Valencia, IBV-CSIC, 46010 Valencia, Spain. 4Howard Hughes and package acetylcholine, genes encoding neuropeptide receptors, Medical Institute, Columbia University Medical Center, New York, NY 10032, USA. various types of ion channels and many others (Altun-Gultekin et al., 2001; Hobert et al., 1997; Wenick and Hobert, 2004). TTX-3 *Author for correspondence ([email protected]) exerts this control through direct binding to a cis-regulatory motif

Received 31 May 2013; Accepted 11 October 2013 shared by all of its target genes. ttx-3 expression is turned on in the Development

422 RESEARCH ARTICLE Development (2014) doi:10.1242/dev.099721 neuroblast that generates AIY and its expression is maintained One of these factors is the POU homeobox gene unc-86, which is throughout the life of the neuron through an autoregulatory feedback required together with ttx-3 to control the identity of the serotonergic loop (Bertrand and Hobert, 2009) to ensure persistent expression of NSM neurons. unc-86 in turn cooperates with the ARID-type its target genes. A number of transcription factors have been transcription factor cfi-1 to control many terminal identity features described in the C. elegans nervous system that display similar of the cholinergic IL2 sensory and URA motor neurons. Our studies broad-ranging effects on the terminal differentiation programs therefore provide further support for the terminal selector concept executed by the neurons in which they are expressed. These and show that, in combination with other regulatory factors, one transcription factors have been called ‘terminal selectors’ (Hobert, factor can serve as terminal selector in distinct neuronal cell types 2008; Hobert, 2011). It is still an open question how broadly the regulating distinct neuronal differentiation programs. terminal selector concept applies throughout the nervous system; that is, how common it is that many distinct and functionally RESULTS unrelated identity features of a specific neuron type are directly co- Expression pattern of ttx-3 in the C. elegans larval and regulated by a transcription factor or a combination of transcription adult nervous system factors. A ttx-3 reporter gene that contains the ttx-3 locus together with a few Here, we investigate the role of ttx-3 in two additional neuron kilobases upstream but no downstream sequences (ttx-3promA::gfp; classes in which it is normally expressed, namely the cholinergic Fig. 1) was previously shown to be continuously expressed in five AIA interneuron class and the serotonergic NSM neuron class. We distinct neuronal cell types: the cholinergic AIY and AIA find in all three neuron classes that there is a common theme of ttx- interneuron classes, the ASI and ADL chemosensory neuron classes 3 function in that it is broadly required to induce many distinct and and a previously uncharacterized neuronal pair in the pharyngeal functionally unrelated terminal identity features of the respective nervous system (Altun-Gultekin et al., 2001). Transient expression neuron class. Yet the downstream targets of ttx-3 in these neuron was observed in the AIN and SMDD neurons at embryonic stages classes are distinct and are determined by the cooperation of ttx-3 (Bertrand and Hobert, 2009). A fosmid reporter construct, which with a distinct set of transcription factors in different neuron classes. contains more than 30 kb surrounding the ttx-3 locus and which

Fig. 1. Expression pattern of the C. elegans ttx-3 LIM homeobox gene. (A) ttx-3 expression constructs and summary of neuronal expression pattern. The promA::gfp and promB::gfp constructs were described previously (Altun-Gultekin et al., 2001; Wenick and Hobert, 2004) and are shown here for comparison only. (B) ttx-3 fosmid expression (wgIs68) in first larval stage animals and in adult animals. D-V, dorsoventral. White asterisks indicate gut autofluorescence. (C) The seventh intron of the ttx-3 locus contains cis-regulatory elements driving reporter gene expression in AIA and NSM neurons. These regulatory

elements do not depend on ttx-3. Expression is shown in adult animals. Development

423 RESEARCH ARTICLE Development (2014) doi:10.1242/dev.099721 rescues the AIY differentiation defect of ttx-3 mutant animals, The AIY interneurons, which have a unipolar axon morphology mirrors the expression of the smaller, locus-restricted reporter similar to that of AIA interneurons in wild-type animals, display construct in the AIY, the AIA, the AIN and the pharyngeal neuron similar morphological defects in ttx-3 mutants (Hobert et al., 1997). class (Fig. 1). Based on position, morphology and colabeling with The expression of terminal identity markers that label several the NSM marker mgl-1::mCherry, we identified the pharyngeal distinct neuron types that are lineally related to AIA is not altered neurons that express ttx-3 as the NSM neuron pair. The NSM (data not shown) (Altun-Gultekin et al., 2001), suggesting that the neurons are serotonergic, neurosecretory cells that are thought to be AIA neuron pair might remain in an undifferentiated state, rather involved in sensing food (Albertson and Thomson, 1976; Harris et than switching to an alternate fate. Based on a more extensive cell al., 2011; Horvitz et al., 1982). fate marker analysis, a similar conclusion was previously drawn There are also notable differences in the expression pattern of the about the fate of the AIY neuron class in ttx-3 mutants (Altun- fosmid reporter and the smaller reporters. First, expression in the Gultekin et al., 2001). Taken together, our fate marker and AIN neurons is maintained throughout development with the fosmid morphological analyses indicate that ttx-3 broadly affects the AIA reporter, whereas it is restricted to embryos with smaller reporters terminal differentiation program. These effects are comparable to the (Bertrand and Hobert, 2009). Second, the expression in amphid previously described broad effects that loss of ttx-3 has on the sensory neurons is markedly different. In larval and adult animals, terminal differentiation of AIY interneurons. the fosmid reporter is expressed in the ASK neuron class, whereas the smaller reporters are expressed in the ADL and ASI sensory A shared cis-regulatory signature of AIA-expressed terminal neurons (Fig. 1). identity features Previous studies have shown that ttx-3 expression in the AIY On a mechanistic level, ttx-3 operates in a distinct manner in the interneuron pair is controlled by a distal initiator element ~1 kb AIA versus AIY neurons since it operates with distinct co-factors upstream of the ttx-3 locus and a maintenance element in the second and through distinct cis-regulatory elements. The co-factor of ttx-3 intron of the ttx-3 locus (Bertrand and Hobert, 2009; Wenick and in AIY, the ceh-10 homeobox gene (Altun-Gultekin et al., 2001), is Hobert, 2004). We find that the expression of ttx-3 in the NSM and not expressed in AIA neurons, and AIA neurons display no AIA is controlled via regulatory elements present in the seventh differentiation defects in ceh-10 null mutants (two markers tested). intron of the ttx-3 locus (Fig. 1). As mentioned above, ttx-3 Moreover, the cis-regulatory motifs through which ttx-3 acts to expression is maintained throughout the life of the AIA and NSM control AIY versus AIA identity are distinct. In the AIY neurons, neurons, but maintained expression of a ttx-3 reporter gene construct ttx-3 acts on its many target genes through a cis-regulatory motif, (ttx-3intron7::gfp; Fig. 1A) in the AIA and NSM neuron types does not termed the ‘AIY motif’, that provides a cooperative binding site for require ttx-3 gene activity (Fig. 1C). a TTX-3–CEH-10 heterodimer (Wenick and Hobert, 2004). Mutation of the AIY motif in a locus that is expressed in AIY and ttx-3 controls the differentiation program of AIA AIA neurons, the cholinergic cho-1 locus, results in a severe interneurons reduction in expression in the AIY interneurons but not in the AIA We focused our analysis of ttx-3 mutants on the cholinergic AIA interneurons (Fig. 3A). interneurons and the serotonergic NSM neurons, which both In the AIA neurons, by contrast, ttx-3 acts through a distinct cis- continuously express ttx-3 throughout their lifetime. We have regulatory signature, which we deciphered through a mutational previously reported that expression of the marker of cholinergic analysis of the cis-regulatory control regions of three AIA- identity, unc-17 (vesicular ACh transporter), as well as the expressed, ttx-3-dependent terminal differentiation genes: mgl-1, ins- expression of an orphan G protein-coupled receptor (GPCR), sra- 1 and cho-1. We generated transgenic animals that express nested, 11, is reduced in the AIA neurons of ttx-3 mutants (Altun-Gultekin shorter versions of these three reporters and identified a 259 bp et al., 2001). We extended this analysis by examining the expression element in the cho-1 promoter, a 74 bp element in the mgl-1 of seven additional markers of terminal AIA fate: the choline promoter and a 68 bp element in the ins-1 promoter that are reuptake transporter encoded by cho-1; the metabotropic glutamate sufficient to direct gfp expression to AIA neurons (Fig. 3A-C). receptor mgl-1; the ionotropic glutamate receptor glr-2; the Examining these elements for common patterns, we noted that all neuropeptides flp-2 and ins-1; the receptor tyrosine kinase scd-2; and these elements contain a shared and phylogenetically conserved the receptor guanylyl cyclase gcy-28d. Each of these markers is G(A/G)ATC motif (Fig. 3D). Mutating this motif in the context of expressed in terminally differentiated AIA interneurons and several any of the three promoters resulted in a reduction of AIA expression of them have previously been implicated in AIA interneuron of the respective reporter (Fig. 3A-C). In the case of mgl-1, two function (Shinkai et al., 2011; Tomioka et al., 2006). The expression G(A/G)ATC motifs are present in the minimal promoter; mutation of each of these seven markers is affected in the AIA neurons of ttx- of either causes an intermediate reduction in reporter gene 3 mutants (Fig. 2). Their expression in other neuron types is expression, and mutation of both motifs results in complete loss of unaffected in ttx-3 mutants, with the exception of two markers that expression (Fig. 3A-C). are also downregulated in NSM neurons (mgl-1, scd-2, as described Since G(A/G)ATC does not match the consensus binding site for below). ttx-3 is likely to act cell-autonomously since the AIA a LIM homeodomain transcription factor such as TTX-3, we also differentiation defects are rescued in transgenic ttx-3 mutant animals examined the minimal reporters for the presence of conserved TAAT that express ttx-3 cDNA under control of the ins-1 promoter motifs, which comprises the core consensus site for LIM (supplementary material Table S1). homeodomain transcription factors (Berger et al., 2008). We indeed AIA neurons remain present in the ttx-3 null mutant, as assessed found several TAAT motifs in the three cis-regulatory modules and by the weak but recognizable expression of some terminal for each of them we identified a TAAT motif that, when mutated, differentiation genes (Fig. 2). However, their normally unipolar affected reporter gene expression in vivo (Fig. 3A-C). These TAAT neurite morphology appears disrupted; ectopic branches can be motifs can be assembled into a larger sequence matrix, TAATTNGA observed to emanate from the cell body and the main neurite (Fig. 3D). In two cases, mutation of the TAATTNGA alone affected

appears blebbed in ttx-3 mutants (supplementary material Fig. S2). reporter gene expression, whereas in the third case (cho-1) a Development

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Fig. 2. ttx-3 affects the terminal differentiation of AIA neurons. (A) Schematic representation of the AIA interneuron pair [reproduced with permission (Altun et al., 2002-2013)]. (B) The expression of terminal differentiation markers of AIA identity is affected in ttx-3 mutants. Reporter gene arrays were crossed into ttx-3(ot22) null mutants. Positions of AIA neurons are outlined (dashed circles). The fraction of animals that show the indicated phenotype is presented in the bar charts. Transgenic arrays are: otIs317 for mgl-1, otIs326 for ins-1, otIs379 for cho-1, otEx4687 for glr-2 and otEx5056 for flp-2 (see Materials and methods for more detail on the arrays; the Ex[gcy-28d::gfp] and Ex[scd-2::gfp] arrays were kindly provided by Takeshi Ishihara). Anterior is up in all panels.

complete loss of expression can only be observed upon protein and probes derived from the mgl-1 and cho-1 locus. We found simultaneous mutation of both the GAATC motif and the TAAT- that TTX-3 is able to bind these sites in vitro (Fig. 3E). Deletion of the containing motif (Fig. 3A). The residual AIA expression of a cho-1 TAAT site that is required for reporter gene expression in vivo resulted reporter construct in which the GAATC motif is mutated, but the in the loss of TTX-3 binding in vitro (Fig. 3E). TAAT motif is left intact, is abolished in ttx-3 mutants (data not The combination of G(A/G)ATC and TAAT motifs might define shown), consistent with ttx-3 operating through the TAAT motif. a cis-regulatory signature that is generally required for gene We examined whether the TAATTNGA motif is indeed a TTX-3 expression in AIA neurons, since we found a combination of these

binding site using gel shift assays with bacterially produced TTX-3 two motifs to be present in the cis-regulatory control regions of the Development

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Fig. 3. Co-regulation of AIA-expressed genes by two cis-regulatory motifs. (A-C) Mutational dissection of the cis-regulatory elements of three AIA- expressed terminal identity markers. (D) Position weight matrix of the two motifs required for AIA expression, based on the motifs from ins-1, cho-1 and mgl-1 and orthologs in other nematode species. Perfect (filled box) and imperfect (stippled box) matches to the two cis-regulatory motifs [blue, G(A/G)ATC; green, TAATTNGA] in other AIA terminal identity markers are shown on the right. (E) TTX-3 binds to cho-1 and mgl-1 regulatory elements containing the HD (TAAT) motif. Deletion of the HD motif abolishes binding. EMSA was performed with 250 nM and 750 nM TTX-3. other four ttx-3-dependent terminal AIA markers (Fig. 3D). Taken involved in monoaminergic transmitter metabolism (Fig. 4A) and together, these data show that AIA identity features are co-regulated identified another NSM-expressed terminal marker, ptps-1 by a shared cis-regulatory signature that is controlled by TTX-3 and (Fig. 4C; supplementary material Fig. S3). In addition to an as yet unknown co-factor. examining these serotonin (5HT)-related markers, we also examined the expression of three metabotropic neurotransmitter ttx-3 controls the terminal differentiation of serotonergic receptors (mgl-1, mgl-3, dop-3), three neuropeptides (nlp-13, flp- NSM neurons 4, nlp-3), a glycoprotein hormone alpha subunit (flr-2) and a We next analyzed the effect of loss of ttx-3 on the terminal receptor tyrosine kinase (scd-2). All of these genes are expressed differentiation program of the serotonergic NSM neurons, a neuron throughout the life of the NSM neurons. As mentioned above, scd- type that has not previously been examined in ttx-3 mutants. Many 2 and mgl-1 are also expressed in AIA neurons, where their terminal identity markers of NSM have been described, including expression is affected by ttx-3. We find that the expression of five the battery of genes that are required to synthesize, package and of these 14 NSM terminal identity markers is either partially or reuptake serotonin: tph-1/TPH (tryptophan hydroxylase), cat- completely eliminated in the NSM neurons of ttx-3 null mutants 4/GTPCH (GTP cyclohydrolase), cat-1/VMAT (vesicular (Fig. 4C, Fig. 5, Table 1). ttx-3 is likely to act cell-autonomously monoamine transporter), bas-1/AAAD (aromatic amino acid since we can rescue the NSM differentiation defects by driving ttx- decarboxylase) and mod-5/SERT (serotonin reuptake transporter) 3 cDNA under the control of a cat-1 promoter fragment, which is (Fig. 4A) (Jafari et al., 2011; Ranganathan et al., 2001; Sze et al., expressed in a subset of monoaminergic neurons of C. elegans 2002). Previous expression analysis of a vesicular glutamate (supplementary material Table S1). transporter, eat-4, suggested that NSM might use the neurotransmitter glutamate (Lee et al., 1999). However, a fosmid- The POU homeobox gene unc-86 also controls NSM identity based eat-4 reporter does not show expression in NSM neurons We recently reported that the effects of the loss of a terminal selector (Serrano-Saiz et al., 2013) (supplementary material Fig. S1A). type transcription factor in dopaminergic neurons can be partially To broaden the spectrum of available terminal markers, we compensated for by other, co-expressed terminal selectors

analyzed the expression of other C. elegans orthologs of enzymes (Doitsidou et al., 2013). Therefore, we considered the possibility that Development

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Fig. 4. The effect of unc-86 and ttx-3 on the serotonergic identity of NSM neurons. (A) The 5HT pathway including tetrahydrobiopterin biosynthesis genes (Deneris and Wyler, 2012). ‘?’ indicates that a unique homolog of SR could not be identified in the worm genome. (B) Schematic representation of the NSM interneuron pair [reproduced with permission (Altun et al., 2002-2013)]. (C) The expression of serotonergic identity features of NSM (dashed circles) is affected in unc-86(n846), ttx-3(ot22) or unc-86(n846); ttx-3(ot22) double-null mutants. Reporter gene arrays were crossed into the respective mutant backgrounds. Transgenic arrays are: zdIs13 for tph-1; otEx4781 for mod-5; otIs225 for cat-4; otEx5280 for ptps-1; otIs226 for bas-1; and otIs224 for cat-1 (see Materials and methods for more detail on the arrays). Images are only shown for mutant genotypes with effects on reporter expression. (D) Serotonin antibody staining. Thirty animals were scored for each genotype. In the double mutant, no animal showed staining in NSM (circled), whereas in the other genotypes all animals showed

staining. Development

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Fig. 5. The effect of unc-86 and ttx-3 on other identity features of NSM neurons. The expression of other identity features of NSM is also affected in unc-86(n846), ttx- 3(ot22) or unc-86(n846); ttx-3(ot22) double-null mutants. Reporter gene arrays were crossed into the respective mutant backgrounds. Transgenic arrays are: vsIs33 for dop-3; otIs317 for mgl-1; otEx5163 for nlp-3; otEx5364 for mgl-3; otEx5163 for nlp-13; otEx5055 for scd-2; and otEx5363 for flr-2 (see Materials and methods for more detail on the arrays). Micrographs are only shown for mutant genotypes with effects on reporter expression. Dashed circles indicate the position of NSM neurons. See also Table 1.

the lack of an impact of ttx-3 loss on nine out of 14 NSM markers we found that the expression of eight is partially or completely could be due to the activity of compensatory terminal selector type eliminated (Figs 4, 5, Table 1). transcription factors. We sought to identify such a factor, focusing To examine whether unc-86 directly affects the expression of on two homeodomain transcription factors previously shown to be these terminal identity features, we analyzed the cis-regulatory expressed in NSM, namely the empty spiracles homolog ceh-2 and control regions of four of them: tph-1, bas-1, cat-1 and cat-4. the POU homeobox gene unc-86 (Aspöck et al., 2003; Finney and Through mutational analysis, we defined small (~200 bp) elements Ruvkun, 1990). We observed no NSM differentiation defects in ceh- that still yielded expression in the NSM neurons (Fig. 6) and, within 2 null animals (data not shown), but we observed striking NSM each of these elements, we identified predicted POU homeodomain differentiation defects in unc-86 mutants. Loss of unc-86 was binding sites (Rhee et al., 1998). We introduced mutations into these previously shown to affect the expression of tph-1 and cat-1 in NSM sites in the context of two loci (tph-1 and bas-1) and found that these neurons, but without effect on 5HT antibody staining (Sze et al., mutations resulted in a loss of reporter gene expression in vivo 2002). Other differentiation features of NSM neurons had not (Fig. 6A,B). Gel shift analysis further confirmed that these POU previously been examined in unc-86 mutants. Upon examining the homeodomain sites indeed bind bacterially produced UNC-86

expression of all 14 markers of NSM fate in unc-86 null mutants, protein in vitro (Fig. 6E). Development

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Table 1. Summary of the effects of ttx-3 and unc-86 null mutants on terminal NSM identity markers Identity feature Function ttx-3(–) unc-86(–) unc-86(–); ttx-3(–) Interaction cat-1 5HT pathway wt dim off Synergism cat-4 5HT pathway dim wt very dim Synergism mod-5 5HT pathway wt wt off Synergism nlp-13 Neuropeptide wt dim off Synergism nlp-3 Neuropeptide wt wt off Synergism flr-2 Transmembrane wt wt dimmer Synergism scd-2 Kinase wt dim off Synergism flp-4 Neuropeptide dim wt stronger expression Synergism 5HT antibody staining Neurotransmitter wt wt off Synergism bas-1 5HT pathway off dim n.d. ? ptps-1 5HT pathway off dim n.d. ? tph-1 5HT pathway wt off n.d. ? mgl-3 GPCR wt off n.d. ? dop-3 GPCR wt off n.d. ? mgl-1 GPCR off wt n.d. ? gfp reporter (or antibody staining): wt, as bright as in wild-type animals; dim, dimmer than in wild type; off, no expression observed. n.d., not determined because single mutant already shows completely penetrant loss of expression. Gray shading indicates presence of defect. unc-86 cooperates with ttx-3 to control NSM identity neurons normally extend a neurite posteriorly toward the nerve ring, We noted that terminal markers of NSM identity that were severely which then bifurcates to form a ventral and a dorsal neurite (Axäng affected in unc-86 mutants tended to be those that were weakly or et al., 2008) (Fig. 4B). We observe that in unc-86(n846) mutants the unaffected in ttx-3 mutants; vice versa, markers unaffected in ttx-3 NSM somas are correctly positioned but there are significant defects mutants tended to be affected in unc-86 mutants (Table 1). Even in outgrowth of the ventral neurite (61% of animals show outgrowth though this observation might simply indicate that unc-86 and ttx-3 defects; n=31). By contrast, in ttx-3(ot22) mutants, the primary act completely independently of one another, we considered the defect observed in ventral neurite outgrowth is the formation of possibility that unc-86 and ttx-3 might collaboratively control NSM aberrant bifurcations (41% of animals show such defects; n=39). identity but that their relative importance may be distinct for Both ttx-3 and unc-86 single mutants also show defects in dorsal different target genes. To investigate this possibility, we examined axon termination [25% (n=32) of ttx-3 mutants and 29% (n=34) of unc-86; ttx-3 double-null mutants and found that markers that are unc-86 mutants]. either partially or unaffected in ttx-3 and unc-86 single mutants are In early larval stages, ventral NSM neurites begin to extend more strongly affected in the double mutant (Figs 4, 5, Table 1). This elaborate arbor structures onto the nerve ring target field (Axäng et also holds for 5HT antibody staining, which is not affected in either al., 2008). These axon arborizations require the NSM-expressed single mutant but completely abrogated in the ttx-3; unc-86 double netrin receptor UNC-40/DCC, which is tightly localized to puncta mutant (Fig. 4D), probably owing to the combined effect that both within the main shaft of the NSM neurite and at the tips of axon genes have on the expression of the 5HT reuptake transporter mod- arbors (Nelson and Colón-Ramos, 2013). These arbor structures 5. As summarized in Table 1, nine of the 15 tested identity features persist into the adult stage and contain presynaptic sites, as assessed (14 reporter genes and 5HT antibody staining) are affected by both with a rab-3 marker (Nelson and Colón-Ramos, 2013). In ttx-3 ttx-3 and unc-86, with effects either visible in both single mutants, mutants, these ultrastructural features are unaffected, but unc-86 or as a non-additive, synergistic effect revealed in the double mutant. mutants display a highly penetrant defect in axon arborization As described below, there are also synergistic effects of ttx-3 and (Fig. 7A,C). Furthermore, unc-86 mutants display defects in the unc-86 on NSM morphology. In six of the 15 cases, either ttx-3 or dynamic regulation of UNC-40 localization (Fig. 7B,D). In wild- unc-86 already has completely penetrant effects (Table 1). Taken type animals, UNC-40::GFP is diffusely distributed at the L1 stage together, these data argue that unc-86 and ttx-3 jointly control and becomes localized to bright puncta in the NSM neurite and at terminal NSM differentiation. The mechanistic basis of the the tips of axon arbors as axons are arborizing at the L4 stage. By cooperation is unclear at present because we have so far not been the adult stage, UNC-40::GFP again becomes diffusely distributed. able to identify functional TTX-3 binding sites in terminal NSM However, a significant fraction of unc-86 mutant NSMs retain a identity marker genes. juvenile-like pattern of UNC-40 localization during the adult stage, To further examine potential interactions of unc-86 and ttx-3, we in which UNC-40::GFP remains localized to bright puncta (Fig. 7B). investigated whether they affect each others expression. We find that We observe synergistic morphological defects in unc-86(n846); continuous expression of unc-86 in NSM neurons depends on unc- ttx-3(ot22) double mutants. Ventral neurites never reach the middle 86 itself [autoregulation of unc-86 was also previously noted of the pharyngeal isthmus and are often truncated immediately (Baumeister et al., 1996)], but not on ttx-3 (supplementary material following the guidance decision to turn posteriorly (100% premature Fig. S1B,C). Vice versa, ttx-3 expression in NSM neurons is not ventral neurite termination, n=18; Fig. 7E). Furthermore, neurites affected in unc-86 or in unc-86; ttx-3 mutants (data not shown). contain large anterior swellings not seen in wild-type animals (33% contain additional anterior swellings, n=18; Fig. 7E). The unc-86 and ttx-3 affect axonal arborization and presynaptic morphological appearance of NSM neurites in unc-86; ttx-3 mutants specializations is reminiscent of the normal morphology of M3 neurons (Albertson Apart from affecting the expression of terminal identity markers, and Thomson, 1976), which are lineally related to NSM (Sulston et loss of unc-86 and ttx-3 also results in specific effects on the al., 1983). M3 neurons are glutamatergic (Lee et al., 1999) and we

morphology of the NSM neurons. During embryonic stages, these indeed find that in unc-86 mutants the vesicular glutamate Development

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Fig. 6. Cis-regulatory analysis of NSM identity specification. (A-D) Dissection of the cis-regulatory elements of four NSM-expressed serotonin pathway genes. All minimal cis-regulatory elements contain predicted POU sites. (E) EMSAs with UNC-86 protein on bas-1 and tph-1 regulatory elements. Mutated POU sites are those that also disrupt reporter gene activity when deleted from the gene contexts of bas-1 and tph-1 (A). EMSA was performed with 10 nM and 30 nM UNC-86. Arrowhead indicates UNC86-bound DNA probe. transporter eat-4 is ectopically expressed in NSMs (supplementary reporter fusions to the unc-17/cha-1 locus and to the choline material Fig. S1A). reuptake transporter cho-1 are expressed in IL2 neurons (Fig. 8A). The expression of these two key markers of cholinergic identity is unc-86 controls terminal differentiation of the cholinergic eliminated in unc-86 mutants (Fig. 8A). Expression of the nicotinic IL2, URA and URB sensory, motor and interneurons acetylcholine receptor subunit des-2 is also lost in the IL2 neurons Apart from our description of unc-86 terminal selector function in of unc-86 mutants (Treinin et al., 1998). the serotonergic NSM neurons, unc-86 had previously been In addition to these cholinergic markers, we examined the described to broadly affect the terminal differentiation program of expression of other genes previously shown to be expressed in IL2 other serotonergic (Sze et al., 2002) as well as glutamatergic neurons, namely the unc-5 netrin receptor, the guanylyl cyclase gcy- (Duggan et al., 1998; Serrano-Saiz et al., 2013) neurons. We asked 19, the kinesin klp-6 and the Notch ligand lag-2 (which is expressed whether unc-86 might affect the terminal differentiation program of in IL2 neurons at the dauer stage) (Leung-Hagesteijn et al., 1992; neurons that use yet another neurotransmitter system. We turned to Ortiz et al., 2006; Ouellet et al., 2008; Peden and Barr, 2005). The the six IL2 sensory neurons that are involved in nictation behavior expression of all of these terminal markers of IL2 identity is (Lee et al., 2012). The IL2 neurons express unc-86 throughout their eliminated in IL2 neurons of unc-86 mutants (Fig. 8A). IL2 neurons lifetime and have been inferred to be cholinergic (Lee et al., 2012). also fail to take up dye in unc-86 mutants (Tong and Bürglin, 2010),

We corroborated the cholinergic identity of the IL2s by finding that suggesting morphological defects. The IL2 neurons are nevertheless Development

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Fig. 7. unc-86 and ttx-3 affect NSM morphology. (A) unc-86(n846) mutant adults display shorter ventral neurites and fewer and shorter axon arbors. mod-5p::gfp (olaEx1446) is used to visualize NSM morphology. (B) UNC- 40::GFP localization (transgene: olaEx1448) remains in a juvenile state in unc-86 mutant animals. White arrows indicate UNC-40::GFP puncta. (C) Quantification of the unc-86(n846) arborization phenotype. Displayed is the fraction of animals with clusters of arbors in the nerve ring region in wild-type and unc-86(n846) animals. The difference between wild type and unc-86 is significant (*P<0.0001). (D) Quantification of the unc-86(n846) UNC- 40::GFP localization phenotype. The fraction of animals with multiple, bright UNC-40::GFP puncta in the nerve ring region is displayed. The difference between wild type and unc-86 is significant (*P=0.0017). Error bars indicate 95% confidence intervals. (E) unc-86(n846); ttx- 3(ot22) double mutants display numerous NSM morphology defects, as visualized with flp- 4::gfp (olaEx1485). In all images, anterior is to the left and ventral down. Asterisks indicate cell bodies (A,B) or additional cell-body-like swellings (E). Brackets denote the nerve ring terminal field where arbors form. White arrows indicate NSM neurites, red arrows and asterisks denote other non-NSM structures. Fisher’s t-test was used for statistical analysis. Scale bars: 5 μm.

generated in unc-86 mutants, as assessed by intact expression of the paper)] is expressed in IL2, URA or URB neurons, unc-86 is likely pan-neuronal marker rab-3 and the pan-sensory marker osm-6 (50 to act with another co-factor in IL2 neurons. cfi-1 is an ARID animals were scored for each marker). transcription factor previously shown to be co-expressed with unc- unc-86 is expressed in two additional cholinergic neuron classes 86 specifically in IL2 and URA neurons (Shaham and Bargmann, in the anterior ganglion besides the IL2 sensory neurons, namely the 2002). Loss of cfi-1 results in ectopic expression of identity markers URA motoneurons [which are synaptically connected to the IL2 for the CEM neuron in IL2 and URA neurons (Shaham and neurons (White et al., 1986)] and the URB interneurons. We found Bargmann, 2002), which prompted us to investigate whether cfi-1 that cholinergic identity was also strongly affected in both URA might also positively control their cholinergic identity. We find that and URB neurons of unc-86(n846) loss-of-function mutants the cholinergic identity of both IL2 and URA neurons is affected in (supplementary material Fig. S4). cfi-1(ky651) loss-of-function mutants, albeit not as strongly as in unc-86 null mutants (Fig. 8A; supplementary material Fig. S4). To unc-86 cooperates with the ARID transcription factor cfi-1 investigate whether unc-86 and cfi-1 genetically interact, we to control IL2 and URA identity examined non-additive synergistic interactions of the two genes Since none of the previously known co-factors of unc-86 [mec-3 for using a hypomorphic unc-86 allele, n848. Animals carrying this

touch neurons (Duggan et al., 1998) and ttx-3 for NSM neurons (this allele show mild IL2 and URA differentiation defects, but in Development

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Fig. 8. unc-86 and cfi-1 control cholinergic IL2 neuron identity. (A) Animals are late L4 or young adults, with the exception of the lag-2::gfp transgenic animals which are dauers. The differential importance of cfi-1 in the dorsal IL2DL/R and ventral IL2VL/R neurons versus the lateral IL2L/R neurons mirrors morphological differences of the ventral versus lateral neurons, with the lateral neurons having a distinct spectrum of synaptic partners (White et al., 1986). See also supplementary material Fig. S4. IL2 schematic reproduced with permission (Altun et al., 2002-2013). (B) Summary of terminal selector combinatorial codes in head ganglia of C. elegans. Colors refer to neurotransmitter identities: green, serotonergic; red, cholinergic; yellow, glutamatergic. Support or blast cells are in gray. combination with the cfi-1(ky651) mutant allele there are strong DISCUSSION synergistic, i.e. non-additive, defects in IL2 and URA differentiation Two main conclusions can be drawn from the data presented in this (Fig. 8A; supplementary material Fig. S4). We conclude that unc-86 paper. First, our data provide general support for the terminal

and cfi-1 cooperate to control IL2 and URA identity. selector concept. Second, our data show that a given transcription Development

432 RESEARCH ARTICLE Development (2014) doi:10.1242/dev.099721 factor can operate as a selector of terminal neuron identity in distinct signature that is composed of two separate motifs located in close neuronal cell types and that this is achieved through cooperation proximity, one a TTX-3 binding site and the other a binding site for with distinct co-factors (summarized in Fig. 8B). In other words, a presumptive TTX-3 co-factor. This is analogous to the situation in individual neuronal cell types use distinct combinatorial codes of the AIY interneuron class, in which TTX-3 and CEH-10 operate terminal selectors, and individual components of the code are reused through a bipartite motif (the ‘AIY motif’) composed of a TTX-3 in distinct combinations in different cell types. and a CEH-10 binding site (Wenick and Hobert, 2004). Genes that The terminal selector concept was initially proposed based on a are expressed in both AIY and AIA neurons (e.g. cho-1) contain a relatively small number of C. elegans transcription factor mutant modular assembly of both the AIY and AIA cis-regulatory signature. phenotypes (Hobert, 2008). In each of these mutant backgrounds, a Similar to the ttx-3-dependent control of the central cholinergic neuronal cell is born and expresses pan-neuronal features but fails interneurons AIY and AIA, the mouse LIM homeobox gene Lhx7 is to adopt neuron type-specific identity features. Importantly, terminal required for the terminal differentiation of cholinergic striatal differentiation is very broadly affected in terminal selector mutants, interneurons (Lopes et al., 2012). As with other terminal selector such that not only functionally linked features (such as enzymes and transcription factors, Lhx7 function appears to be continuously transporter in a neurotransmitter synthesis/transport pathway), but required to maintain cholinergic identity. Co-factors that operate also seemingly completely independent differentiation features that together with Lhx7 are currently not known. Lhx7 is expressed in have no obvious biochemical connection (e.g. sensory receptors, many other neurons in the CNS. It will be interesting to determine neuropeptides and ionotropic neurotransmitter receptors) fail to be whether Lhx7 also operates as a terminal selector in these other expressed. That the removal of an individual transcription factor neuron types. results in such broad defects could not necessarily be assumed since ttx-3 activity is not restricted to cholinergic neurons. We find that transcriptomic approaches generally show that individual cell types ttx-3 is also a key regulator of serotonergic neuron identity. The expresses several dozen transcription factors (e.g. Etchberger et al., activity of ttx-3 in the serotonergic NSM neuron class is, however, 2007). This could be interpreted to mean that the identity features distinct from that of AIA and AIY. Whereas the expression of of a neuron are regulated in a piecemeal manner, rather than being several NSM-expressed effector genes is completely eliminated in ‘mastered’ by a single transcription factor or a small combination ttx-3 mutants, the expression of some effector genes is only partially thereof (Hobert, 2011). Two major questions raised by the terminal affected or not affected at all. In cases in which only partial or no selector concept were how broadly it applies to different cell types effect was observed, joint removal of another homeobox gene, unc- in the C. elegans nervous system and how it applies to transcription 86, resulted in much stronger or complete loss of effector gene factors expressed in distinct neuron types. expression. Vice versa, the expression of effector genes that are Here, we have shown that the terminal differentiation programs unaffected in expression in unc-86 mutants is lost in either ttx-3 of very distinct neuron types – a cholinergic interneuron (AIA), a mutants or in the ttx-3; unc-86 double mutant. Taken together, serotonergic sensory/motor neuron (NSM) and cholinergic sensory elimination of both of the POU/LIM homeobox genes unc-86 and and motor neuron classes (IL2 and URA) – are controlled by distinct ttx-3 has profound effects on NSM identity, paralleling the profound combinatorial codes of transcription factors. These factors regulate effect that another POU/LIM homeobox combination (unc-86 and many distinct identity features of these distinct neuron types, mec-3) has on touch neuron differentiation (Duggan et al., 1998). ranging from neuropeptides to neurotransmitter synthesis pathway How unc-86 and ttx-3 interact to control NSM differentiation is genes to neurotransmitter receptors and other signaling molecules. currently unclear. Both genes are continuously expressed in NSM In the case of the cholinergic AIA interneuron, we found that the neurons, but do not regulate the expression of each other. Based on expression of every tested terminal differentiation marker is affected the synergistic nature of the effect of joint ttx-3 and unc-86 removal in ttx-3 mutants. Since the available AIA marker collection on the expression of some target genes (no or limited effect in single essentially represents a random snapshot of terminal markers that mutants, complete loss in double mutant), we propose that both characterize AIA identity, one might extrapolate the regulatory transcription factors act jointly on common target gene promoters. impact of ttx-3 on each one of these genes to the many hundreds, if For some target genes, the loss of one regulatory factor can be not thousands, of genes that are expressed in AIAs, such that ttx-3 completely or partly compensated for by the other regulatory factor; is likely to affect a very large number of them. The estimated very in other cases, such compensation is not possible. unc-86 and ttx-3 broad effect of ttx-3 on AIA identity is consistent with what we might therefore not always act in a strict cooperative sense, but observed for the cholinergic AIY interneuron, in which ttx-3 rather act independently on target gene promoters. There is already mutation also affects the expression of all known identity features a notable precedent for such a mechanism, as we recently found that (Altun-Gultekin et al., 2001; Wenick and Hobert, 2004). Even a combination of three different transcription factors controls though both neuron types have similar morphologies, are dopaminergic neuron identity. For some target genes, individual cholinergic, and are directly postsynaptic to various sensory neurons, transcription factor mutants display very limited effects, but double AIY and AIA have different functions (Hobert et al., 1997; Shinkai mutants strongly affect target gene expression (Doitsidou et al., et al., 2011; Tomioka et al., 2006), connect to a different spectrum 2013). In the case of NSM, we cannot however rule out the of synaptic partners (White et al., 1986) and express distinct gene possibility that some genes are exclusively regulated by unc-86 batteries. Yet, in both cases, ttx-3 very broadly affects the whereas others are exclusively regulated by ttx-3. differentiation of each neuron type. Apart from demonstrating ttx-3 terminal selector function in The distinct target gene specificities of ttx-3 in AIA and AIY distinct neuron types, we have also shown here that the POU neurons can be explained by neuron type-specific co-factors and by homeobox gene unc-86 can similarly act as a terminal selector in ttx-3 acting through distinct cis-regulatory motifs. AIY-expressed distinct neuron types. A role of unc-86 in the differentiation of genes display a characteristic cis-regulatory signature that is serotonergic and glutamatergic touch neurons has been described recognized by a combination of the TTX-3 and CEH-10 previously (Desai et al., 1988; Duggan et al., 1998; Sze et al., 2002; homeodomain proteins (Wenick and Hobert, 2004). As we have Serrano-Saiz et al., 2013). We show here that unc-86 also controls

shown here, AIA-expressed genes share a distinct cis-regulatory the terminal differentiation programs of three distinct cholinergic Development

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neuron types. Two of these cholinergic neuron types are synaptically N.F.); a VAL i+d Fellowship from Generalitat Valenciana (to C.L.-F.); and a connected and form a simple sensory-to-motor circuit (White et al., European Research Council Starting Grant (to N.F.). N.F. is a National Alliance for 1986). The role of unc-86 in controlling cholinergic IL2 sensory Research on Schizophrenia and Depression (NARSAD) Young Investigator. O.H. is an Investigator of the Howard Hughes Medical Institute. Deposited in PMC for neuron specification is reminiscent of, and might even be release after 6 months. homologous to, the function of the POU homeobox gene acj6 in controlling expression of the cholinergic gene locus in Drosophila Supplementary material olfactory neurons (Lee and Salvaterra, 2002). The ARID-type Supplementary material available online at transcription factor cfi-1 cooperates with unc-86 to control the http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.099721/-/DC1 cholinergic identity of IL2 and URA neurons. Although neuronal References differentiation functions have been reported for the cfi-1 homolog Albertson, D. G. and Thomson, J. N. (1976). The pharynx of Caenorhabditis elegans. dead ringer (retained – FlyBase) in Drosophila (Ditch et al., 2005), Philos. Trans. R. Soc. B 275, 299-325. the functions of vertebrate orthologs (Arid3 genes) in the nervous Altun, Z. F., Herndon, L. A., Crocker, C., Lints, R. and Hall, D. H. (eds) (2002-2013). WormAtlas, http://www.wormatlas.org. system remain to be explored. Altun-Gultekin, Z., Andachi, Y., Tsalik, E. L., Pilgrim, D., Kohara, Y. and Hobert, O. (2001). A regulatory cascade of three homeobox genes, ceh-10, ttx-3 and ceh-23, controls cell fate specification of a defined interneuron class in C. elegans. MATERIALS AND METHODS Development 128, 1951-1969. Strains and transgenes Aspöck, G., Ruvkun, G. and Bürglin, T. R. (2003). The Caenorhabditis elegans ems For a list of strains and transgenes and notes on their generation see class homeobox gene ceh-2 is required for M3 pharynx motoneuron function. supplementary material Table S2. Development 130, 3369-3378. Axäng, C., Rauthan, M., Hall, D. H. and Pilon, M. (2008). Developmental genetics of the C. elegans pharyngeal neurons NSML and NSMR. BMC Dev. Biol. 8, 38. Serotonin antibody staining Baumeister, R., Liu, Y. and Ruvkun, G. (1996). Lineage-specific regulators couple Young adult animals were fixed in 4% paraformaldehyde overnight and then cell lineage asymmetry to the transcription of the Caenorhabditis elegans POU gene treated with 5% β-mercaptoethanol overnight followed by 1000 units/ml unc-86 during neurogenesis. Genes Dev. 10, 1395-1410. collagenase (Sigma-Aldrich) treatment. Rabbit anti-serotonin whole serum Berger, M. F., Badis, G., Gehrke, A. R., Talukder, S., Philippakis, A. A., Peña- Castillo, L., Alleyne, T. M., Mnaimneh, S., Botvinnik, O. B., Chan, E. T. et al. (Sigma-Aldrich, S5545) was used at 1:100 dilution. Worms were then (2008). Variation in homeodomain DNA binding revealed by high-resolution analysis washed and incubated with Alexa Fluor 555 donkey anti-rabbit IgG (1:1000; of sequence preferences. Cell 133, 1266-1276. Life Technologies, A-31571). Bertrand, V. and Hobert, O. (2009). Linking asymmetric cell division to the terminal differentiation program of postmitotic neurons in C. elegans. Dev. Cell 16, 563-575. Clark, S. G. and Chiu, C. (2003). C. elegans ZAG-1, a Zn-finger-homeodomain Cis-regulatory analysis protein, regulates axonal development and neuronal differentiation. Development DNA sequences were subcloned into pPD95.75 expression vector 130, 3781-3794. (Addgene). For some smaller constructs, PCR products were directly Deneris, E. S. and Wyler, S. C. (2012). 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Mouse brain organization revealed through direct genome-scale TF expression analysis. Science 306, 2255-2257. Competing interests Greer, E. R., Perez, C. L., Van Gilst, M. R., Lee, B. H. and Ashrafi, K. (2008). Neural and molecular dissection of a C. elegans sensory circuit that regulates fat and The authors declare no competing financial interests. feeding. Cell Metab. 8, 118-131. Harris, G., Korchnak, A., Summers, P., Hapiak, V., Law, W. J., Stein, A. M., Author contributions Komuniecki, P. and Komuniecki, R. (2011). Dissecting the serotonergic food signal F.Z. and O.H. initiated the study. A.B. and P.G. performed the analysis of the IL2 stimulating sensory-mediated aversive behavior in C. elegans. PLoS ONE 6, neurons; A.B. performed analysis of the URA and URB neurons; C.L.-F., M.M. and e21897. N.F. undertook the mutational analysis of the serotonergic pathway promoters; Hirota, J. and Mombaerts, P. (2004). The LIM-homeodomain protein Lhx2 is required J.C.N. and D.A.C.-R. performed and supervised the morphological analysis of the for complete development of mouse olfactory sensory neurons. Proc. Natl. Acad. NSM neurons; N.A. and R.S.M. performed and supervised the gel-shift analyses. Sci. USA 101, 8751-8755. F.Z. performed all other experiments. O.H. wrote the paper. Hobert, O. (2008). Regulatory logic of neuronal diversity: terminal selector genes and selector motifs. Proc. Natl. Acad. Sci. USA 105, 20067-20071. Hobert, O. (2011). Regulation of terminal differentiation programs in the nervous Funding system. Annu. Rev. Cell Dev. Biol. 27, 681-696. This work was funded by the National Institutes of Health [R01NS039996-05 and Hobert, O. and Ruvkun, G. (1998). A common theme for LIM homeobox gene R01NS050266-03 to O.H.; R01NS076558 to D.A.C.-R.; and R01NS070644 to function across phylogeny? Biol. Bull. 195, 377-380. 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